Tinnea gombea (Lamiaceae), a new species from the Sudanian savanna region, Nigeria based on integrative evidence

Tinnea gombea, endemic to the Sudan savanna grasslands in northern Nigeria, is described and illustrated. We used integrative evidence from morphological characters, ecology and molecular phylogenetic data. The new species is morphologically and ecologically similar to T. barteri and T. aethiopica, but can be readily delimited from these taxa by unique characters including a subshrub growth habit, leaves alternate to subopposite, blades lanceolate, apically acuminate, inflorescences raceme, bearing solitary flowers in upper leaf and bract axils, lilac to purplish dusky flowers and the inflated fruits dehiscent. The distribution and habitat of T. gombea are also distinctive, being restricted to the Sudan savanna, while the two most similar species are widespread in tropical Africa. Additionally, molecular phylogenetic assessments using nrITS and chloroplast trnL-F, matK and rbcL support the placement of T. gombea as a distinct species. Tinnea gombea is here assessed as Critically Endangered due to its small population size and restriction to a small area lacking conservation prioritization.


Introduction
Tinnea Kotschy ex Hook.f, is a genus in the family Lamiaceae, subfamily Scutellarioideae, comprises 19 species native to the African continent [1,2]. Species of the genus are found in the west, central and east tropical and southern subtropical regions occupying the savanna scrubs and the great Guinean forest. All species are narrow-ranged endemics except the widespread T. aethiopica Kotschy ex Hook.f. and T. barteri Gürke that extend from coastal areas of east Africa to tropical west and central Africa and to subtropical southern Africa [2]. The subtropical regions of southern Africa recorded the highest species diversity [3].
Tinnea species are erect annual or perennial herbs, suffrutices or shrubs arising from woody rootstock with quadrangular or terete stem transverse sections, covered with simple hairs; leaves typically simple, opposite or ternate, covered with unicellular to glandular trichomes on both adaxial and abaxial surfaces, with glands; bracts leaf-like but smaller; inflorescence racemose, in axils of upper leaves and bracts or forming terminal spikes; flowers typically pedicellate, solitary or cymose, upper part of the calyx 2-lipped, short and flat but 3-lipped, broad and spoon-shaped at lower side; becoming enlarged; and nutlets inflated, with conspicuous ridge of wings on the dorsal side [2].
The placement of the genus in Scutellarioideae was based on tuberculate corollas, exserted stamens and inflated fruits [1][2][3], and later was confirmed by phylogenetic studies of Li et al. [4] and Zhao et al. [5]. Robyns and Lebrun [1] separated the genus into two sections based on the floral arrangements namely, section Sparsiflora with solitary or 2-3-cymes flowers not forming distinct clusters and section Spicata with elongated spikes in axils of upper leaves and bracts or forming clusters at the terminal head. Section Spicata was further delimited into two subsections namely Scariosae (with two series) and Membranaceae based on calyx characters. The calyx in the former being scarious and the latter membranous. Although Robyns and Lebrun's [1] monograph is the most comprehensive taxonomic treatments of Tinnea, the species delimitation and classification were completely artificial [5]. Therefore, the modern tools of integrative phylogenetics are required to test this hypothesis.
The rapid increase of genomic sequence data has made phylogenetics an indispensable tool for identification and classification of plants. The last two decades have witnessed growth in molecular phylogenetic studies in Lamiaceae [e.g. [6][7][8], at the subfamilial and tribal level [e.g. [9][10][11][12] as well as at generic levels [e.g., [13][14][15]. Although Li et al. [4] and Zhao et al. [5] included Tinnea zambesiaca Baker and T. aethiopica respectively in their molecular phylogenetic studies of Lamiaceae, assessments of species' phylogenetic relationships within the genus Tinnea is lacking. According to Li et al. [4], the genus Tinnea is a close sister to Scutellaria L. The sampling of Tinnea species in these studies, however, was insufficient to allow assessing the monophyly of the genus and to contribute in understanding evolutionary relationships within Tinnea based on molecular phylogenetic analyses.
During field trips for the preparation of a Field Guide to Herbaceous Plants of the Savanna in Gombe State, Sudan savanna, northeastern Nigeria, we encountered noteworthy Tinnea populations on farmlands in Akko Local Government Area. However, we were unable to key the Tinnea collections to the descriptions from the Flora of West Tropical Africa [15], Flora of Tropical Africa [16], a handbook of West African Weeds [17], Flora Zambesiaca [18], African Plant Database [19] and Plants of the World Online [2]. After a detailed examination of the specimens, we hypothesized that these populations represent an undescribed species. Herein, we formally describe and illustrate this species new to science using morphological, ecological and molecular evidence.

Morphological study
Morphological assessments were based on herbarium specimens and our field collections. Further, protologues of all published names in the genus Tinnea from Africa and all relevant taxonomic literature [1-3, 15, 16, 20] were consulted and reviewed. To delimit the potential new taxa from closely similar species, morphological variations were compared with herbarium (FHI, GSUH, K, S; acronyms follow [21]) specimens including type materials on JSTOR [22] and POWO [2]. A dissecting microscope (Leica GZ4) or stereomicroscope (Leica S9i) fitted with digital camera (Nikon DS-5M) and eyepiece micrometer were used to measure and photograph stems, leaves, inflorescences, flowers and fruits.

Phylogenetic study
To assess the molecular phylogenetic placement of the new species within the genus Tinnea, three chloroplast DNA markers (matK, rbcL, trnL-F) and the nuclear ITS were used following [6,12,23]. Whole genomic DNA of the new species was extracted from 0.2 g of silica-gel dried leaves using the standard CTAB protocol [24] as amended by [23]. For detailed information on oligonucleotides and DNA markers, polymerase chain reaction mix, amplification thermal profiles and sequencing methods used, see our previous study [23]. Other sequences available for the genus Tinnea from other authors were downloaded from GenBank (https://www.ncbi. nlm.nih.gov/nuccor). Two species in the closest genus Scutellaria L. (Lamiaceae), S. lateriflora L. and S. indica Blume were selected as outgroups based on the Lamiaceae phylogenetic relationship hypothesized by [12] and [13]. Newly generated sequences were deposited on Gen-Bank (see bold accession numbers, Table 1). Detailed information on accessions sampled for this study are provided in Table 1.
Five sequence datasets were generated: three chloroplast DNA datasets including, matK, rbcL, trnL-F, the nuclear ITS dataset and concatenated datasets (including all four DNA markers). Prior to the concatenation, the individual dataset was aligned using MAFFT Online service [25,26] and manually checked and adjusted in BioEdit version 7.2.6 [27]. The individual and concatenated datasets were phylogenetically analyzed using the Bayesian Inference and Maximum Likelihood approaches using MrBayes version 3.2.6 x64 on XSEDE [28] and RAxML [29] as implemented in CIPRES cluster [1][2][3], respectively. For the nucleotide model substitutions of each DNA marker, the package jModelTest v.3.7 [30,31] under the Akaike and information criteria (AIC) was independently selected. Thus, GTR+I+ Γ for ITS, GTR+ Γ for matK and GTR+I for rbcL and trnL-F. For details on the BI and ML analyses, refer to the phylogenetic work in [23] except that the Markov Chain Monte Carlo analyses for both BI and ML were run for 50,000,000 generations and sampling a tree at every 1,000th generation. The first 0.25 of sampled generations were discarded as "burn-ins" and the 50% majority-rule consensus tree was obtained from the remaining trees.

Distribution map and conservation assessments
The distribution map was generated from specimen localities obtained from our field collections and herbarium voucher information using the naijR package version 0.4.0 as implemented in R version 4.2.0 [32]. Conservation status of a species is substantiated if one of the Bendisks 057 ---https://doi.org/10.1371/journal.pone.0280550.t001

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criteria A-E outlined in the guidelines of IUCN is established [33,34]. Hence, the conservation status of the new species was assessed based on Criteria B (based on the area of occupancy and extent of occurrence), C and D (restricted, with a very small population size of <2,500 individuals and known only from the type locality) of the IUCN, [34]). The Geospatial Conservation Assessment Tool (GeoCAT; [35]; http://geocat.kew.org/) was used to quantify the Area of occupancy (AOO) and the extent of occurrence (EOO) with the default 2 × 2 km 2 size following [36].

Nomenclature
The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies. In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories PubMed Central, LOCKSS.

Results
The statistic properties of the ITS, trnL-F, matK, rbcL and combined datasets are presented in Table 2. Even though the aligned sequence length for ITS was shorter than those of trnL-F and matK, the percentage of parsimony informative sites was significantly higher (32.1% versus 19.5% and 2.0% respectively). Further, the number of variable sites and consistency index recovered for each dataset reflect the lower phylogenetic signal in the chloroplast datasets versus the ITS. There are a number of shared alignment features between Scutellaria and Tinnea, e.g. a deletion (gap) in ITS, aligned position 103-132 that is not observed in any other Tinnea species. The already strong support values for a relationship between these two genera would have increased if gap coding had been used.
The molecular phylogenetic relationships among the Lamiaceae taxa as indicated in the ITS and chloroplast trees were highly congruent, although less resolved in the latter. Further, the tree topologies obtained from BI and ML analyses were congruent. Thus, only the 50% majority-rule consensus tree obtained from the Bayesian analysis is here presented (Fig 1). Using Scutellaria to root the tree, two strongly supported main clades are recovered: the first containing the outgroup Scutellaria species and the second containing the remaining Tinnea species. The monophyly of Tinnea was strongly supported as revealed by both bootstrap (BS) and  Fig 2), T. gombea is characterized by erect growth habit, 20-50 cm tall, unbranched to lax branching pattern, from taproot system. The stems are single or 2-3 in number, terete, light green or glaucous appearance due to woolly hairs covering the stem and changes to tan with age. The leaves are alternate or subalternately arranged, attached to the stem by subsessile petioles, blade lanceolate, about 5 × 3 cm, covered with simple trichomes and conspicuous veins on both adaxial and abaxial surface, with abaxial surface usually light green, adaxial surface green basally obtuse, margins entire, apex acuminate. The racemose inflorescences have solitary flowers in upper leaf and bract axils, flowers with short pedicels, lilac to purplish grey, upper corolla lips shorter than the lower lips, with yellowish white

Discussion
A detailed morphological comparison of the new species and other allied species of Tinnea was conducted (Table 3). Tinnea gombea is similar to T. barteri and T. aethiopica (Table 3; [15]). However, Tinnea gombea can be distinguished from both by a series of morphological traits such as having shorter petioles (usually to about 0.5 mm), leaves alternate to subalternately arranged, blade lanceolate, apically acuminate, shorter pedicels (usually <5 mm), lilac to purplish dusky flowers and fruits being dehiscent (versus both T. aethiopica and T. barteri having petioles ca. 5 mm long, orbicular or rarely long obovate leaves, apically apiculate and mucronate respectively, longer pedicels (usually >5 mm), purplish or reddish brown to blackish purple flowers and fruits being indehiscent). Tinnea gombea best fits into Robyns and Lebrun's [1] section Sparsiflora. It shares the solitary flowers in the axils of the upper leaves with other species in this section. However, some characters in T. gombea overlap with species in section Spicata too. For example, elongated racemose inflorescences and flowers solitary or grouped as 2-5-cymes in bract axils are typical of species in section Spicata [1]. Although the floral arrangements were the main diagnostic character of sections in Tinnea [1], there were overlaps of these characters in species of the two sections.
Molecular phylogenetic analyses placed T. gombea as sister to a clade (Fig 1) comprising T. barteri and three southern African taxa (T. zambesiaca, T. gracilis, T. rhodesiana). The results of our phylogenetic analyses are generally congruent with the morphological and biogeographic patterns but do not support the infrageneric classification scheme proposed by [1].
In habitat, as for T. barteri and T. aethiopica, T. gombea also occurs in the savanna grasslands and woodlands of tropical Africa [1][2][3]. Within the savanna grasslands, however, T. gombea is restricted to the ecotones of loamy soil and rocky outcrops of the Sudanian savanna (Fig  3). It is noteworthy that these two species (T. aethiopica and T. barteri) were the only Tinnea reported previously in west tropical Africa [15].
Thus, the integrative taxonomic evidence suggested by Sangster [37], here based on morphological variations (Figs 2 and 4; [1]), ecology and molecular phylogenetic position (Fig 1) strongly support the recognition of T. gombea as an evolutionarily independent and distinct taxon of a new species in Tinnea. Given the topology of our phylogenetic tree and the strong

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Tinnea gombea, a new species from the Sudan savannah

Etymology
Tinnea gombea is named after its type locality, Gombe State, Nigeria.

Phenology
Tinnea gombea flowers from August to September and fruiting between September and October.

Distribution and habitat
Tinnea gombea is endemic to the Sudanian savanna and is currently known from Gombe State (Fig 3). Apparently, the species is uncommon within its area of occurrence in grasslands and woodlands of Sudanian savanna. Usually found on abandoned farmland together with various annual herbs and with perennial shrubs such as Spermacoce L. species, Oldenlandia corymbosa L., Eragrostis tremula Hochst., Physalis angulata L. and Vernonia ambigua Kotschy & Peyr. It usually grows at an elevation of about 620 m above sea level.

Conservation assessments
Tinnea gombea is known from the type locality only. We recorded <100 mature individuals in each of the three subpopulations. We have made a concerted effort to sample the species from other potential localities within and around abandoned farmlands in the Sudan savanna but were unsuccessful. Therefore, further botanical surveys for possible localities for this species is recommended. At present, the area of occupancy (AOO) of 0.5 km 2 and an extent of occurrence (EOO) of 5.00 km 2 were estimated for the known subpopulations. We project a continuous decline in the AOO, EOO, number of subpopulations and number of mature individuals for T. gombea given the level of accelerated urbanization toward its area of occurrence. Further, flooding and agricultural activities (crop cultivation and overgrazing) are critical threats to this species. Hence, preliminarily, the red list status of Critically Endangered (CR; B1, B2 (b, c), C2 (a, b) and D) under the categories and criteria B-D of the IUCN [34] guideline is assigned to T. gombea.